José Guilherme Neves
José Guilherme Neves
ENERGIA LIVRE DE SUPERFÍCIE DO ESMALTE
DENTÁRIO TRATADO COM HEXAMETAFOSFATO DE
SÓDIO, CÁLCIO E FOSFATO
José Guilherme Neves
José Guilherme Neves
ENERGIA LIVRE DE SUPERFÍCIE DO ESMALTE
DENTÁRIO TRATADO COM HEXAMETAFOSFATO DE
SÓDIO, CÁLCIO E FOSFATO
Dissertação apresentada à Faculdade de Odontologia da
Universidade Estadual Paulista “Júlio de Mesquita Filho”,
Campus de Araçatuba, para obtenção de título de Mestre em
Ciência Odontológica - Área de Concentração: Biomateriais.
Orientador: Prof. Titular Alberto Carlos Botazzo Delbem
Coorientadora: Profa. Dra. Marcelle Danelon
José Guilherme Neves Catalogação na Publicação (CIP)
Diretoria Técnica de Biblioteca e Documentação – FOA / UNESP
Neves, José Guilherme.
N518e Energia livre de superfície do esmalte dentário tratado com hexametafosfato de sódio, cálcio e fosfato / José Guilherme Neves. - Araçatuba, 2016
64 f. : il. ; tab. + 1 CD-ROM
Dissertação (Mestrado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba
Orientador: Prof. Alberto Carlos Botazzo Delbem Coorientadora: Profa.Marcelle Danelon
1. Fosfatos 2. Esmalte dentário 3. Energia I. Título
Black D15
José Guilherme Neves
Dados Curriculares
José Guilherme Neves
Nascimento 09.03.1992 – Piracicaba-SP
Filiação
Rodinei José Neves
Fernanda Aparecida Cabral Neves
2011/2014 Curso de Graduação em Odontologia pelo Centro
Universitário Hermínio Ometto- Uniararas
2015/2016 Desenvolvimento de Projeto de Mestrado com auxílio da
Coordenação de Pessoal de Nível Superior - CAPES
Associações CROSP - Conselho Regional de Odontologia de São Paulo.
José Guilherme Neves
COMISSÃO EXAMINADORA
DISSERTAÇÃO PARA OBTENÇÃO DO GRAU DE
MESTRE
Prof. Titular Alberto Carlos Botazzo Delbem – Orientador, Professor Titular do Programa de Pós-Graduação em Ciência Odontológica, Departamento de Odontologia Infantil e Social, Disciplina de Odontopediatria da Faculdade de Odontologia - Araçatuba, UNESP - Universidade Estadual Paulista Júlio de Mesquita Filho, Araçatuba.
Prof. Dr. Douglas Roberto Monteiro – Professor Permanente do Programa de Pós-Graduação em Ciência Odontológica, Departamento de Odontologia Infantil e Social. Área de concentração Biomateriais- Faculdade de Odontologia - Araçatuba, UNESP - Universidade Estadual Paulista Júlio de Mesquita Filho, Araçatuba.
José Guilherme Neves
José Guilherme Neves Primeiramente gostaria de dedicar este trabalho às pessoas que mais
me apoiaram desde o dia do meu nascimento.
Meus pais,
Rodinei e Fernanda
.
Muitas vezes me faltam palavras para descrever tudo o que eu sinto
por vocês. Talvez gratidão e amor possam traduzir apenas uma parte do
que eu sinto. Gratidão por vocês terem me concebido, e desde então não
medirem esforços para sempre nos dar o melhor, muitas vezes deixando
seus próprios limites de lado.
Nesse período o qual permaneci um pouco distante de vocês, me fez
ver o quanto o amor entre nós é forte, o quanto cada momento perto de
vocês é especial. Me sinto amado por vocês em todos os momentos, em
todos os gestos, e em todas as palavras.
Eu aprendo muito com vocês, com o caráter de vocês, com a forma
com a qual vocês lidam com as situações. Vocês são as pessoas mais
integras, honestas e com o coração voltado em fazer a vontade de Deus
que eu já conheci.
Obrigado por sonharem comigo esse grande sonho! Vocês podem
ter a certeza que um dia eu irei retribuir tudo isso!! Eu irei honrá-los até
meus últimos dias!!
Amo vocês com todas as forças
“Quero consagrar meu lar a Ti, o nosso futuro para te servir.
Com toda minha força e entendimento, quero dedicar o meu lar a
José Guilherme Neves
José Guilherme Neves
A Deus
Pai, como sou grato ao Senhor por me escolher desde o ventre da
minha mãe. Por me dar o dom da vida, e me fazer crescer em graça e
estatura. Obrigado por confiar em mim talentos os quais eu sempre usarei
para louvar e glorificar o Teu nome.
Não há nada que eu anseie mais em minha vida, do que estar no
centro da Tua vontade. Em fazer com que os Teus planos em minha vida
sejam cumpridos conforme a Tua vontade. Eu sou um servo sempre pronto
a Te servir, aonde quer que eu esteja.
Sou grato por me amar incondicionalmente, me proteger, me guiar,
me confortar e me fazer entender que as coisas ruins são para o
amadurecimento e formação do meu caráter.
Os meus lábios sempre proclamarão Tua verdade, o Teu amor e Tuas
obras.
Eu te amo mais do que o meu entendimento, e com a certeza de que
o Senhor está me guiando em todos os lugares onde eu estiver.
Ao Senhor Jesus toda a glória, louvor, honra e minha adoração.
Ao meu irmão Vinicius,
Obrigado por sempre estar comigo, sempre me apoiando em todos
os meus sonhos e projetos. Você é um grande espelho para mim! Conte
José Guilherme Neves
A minha sobrinha Luiza,
A você que trouxe ainda mais vida, alegria e amor para as nossas
vidas, o meu agradecimento! Você reafirmou o meu amor pela minha
profissão e me fez entender melhor o seu mundo. Ouvir sua voz, e te ver
crescer me faz acreditar em um futuro melhor.
Talvez hoje você não entenda as palavras, as quais estou escrevendo,
mas o dia que você tiver a oportunidade de ler, saiba que o meu amor por
você é incondicional e que você me enche de luz e amor!
Eu te amo minha amiguinha...
A minha querida e amada avó Neusa Ricci,
Como sou grato à senhora por me amar com a força, a qual me ama!
A senhora me conhece como ninguém, conhece os meus anseios, sonhos,
insegurançass e medos. A senhora é minha confidente, minha melhor
amiga, em quem eu confio tudo! Se eu pudesse fazer um pedido a Deus, eu
pediria que a senhora fosse para sempre minha,
Como nossas tardes são especiais, sempre regadas de longas
conversas, risadas, e com a senhora me ouvindo cantar com aquele olhar
de admiração.
Obrigado por todos os dias interceder por mim perante Deus e os
José Guilherme Neves
A minha grande parceira da vida Larissa Leibholz,
Que sorte a nossa das nossas vidas terem se cruzado! Ter você ao
meu lado me faz sentir que eu estou em paz, que estou no meu porto
seguro. Te amei desde a primeira vez que eu te vi, te amo hoje, e te amarei
para sempre!
A minha irmã do coração Vanessa Marques,
Nossa história começou quando na primeira aula do mestrado
decidimos fazer dupla em alguns seminários. Mal sabíamos nós, que Deus
tinha nos colocado ali para que algo muito maior fosse construído. Nos
tornamos cumplices, amigos e irmãos.
Em momentos de alegria, você foi quem eu corri contar e comemorar
o momento festivo. Em momentos de tristeza, dor e inseguranças você foi
quem me acolheu com todo amor e carinho, enxugou minhas lagrimas e
me fez ver que aquele seria apenas um dia ruim e que um novo dia
amanheceria com alegria.
Como sou grato a Deus por ter você em minha vida, e por poder falar
que hoje, você é parte da minha família. Obrigado por todo cuidado, carinho
e atenção que dedicou a mim. Voce foi parte fundamental dessa grande
etapa da minha vida.
Meu eterno amor, carinho e gratidão a você por tudo que fez, faz e é
para mim!
José Guilherme Neves
A minha grande amiga Mayra,
Agradeço a Deus por ter te encontrado nessa fase tão importante de
nossas vidas. Compartilhamos juntos de momentos muito alegres, cheio de
risadas, e é disso que eu quero lembrar. Lembrar do seu astral, da sua
energia, sempre vendo as coisas de forma mais leve, e com um jeito lindo
de se ver a vida. Obrigado por me acolher sempre que precisei, e por
sempre me mostrar que as coisas no final sempre dariam certo.
Você é muito especial para mim, meu eterno carinho e gratidão a
você!
Eu amo você, obrigado por tudo!
Ao meu Orientador,
Prof. Titular. Alberto Carlos Botazzo Delbem
Gostaria de agradecer ao senhor pela grande oportunidade de
realizar esse sonho. Agradecê-lo por me receber e confiar em minha
capacidade para desenvolver parte do seu estudo.
Foi uma honra trabalhar com um pesquisador como o senhor, um
profissional de extrema excelência, e com um conhecimento o qual não sei
se seria possível listar.
Nesse breve período em que convivemos, fiquei ainda mais inspirado
a seguir o meu sonho de me tornar pesquisador, e por essa razão, serei
eternamente grato. Ser seu orientado foi uma honra e um prazer imenso. Ao
José Guilherme Neves
A minha Coorientadora,
Professora Doutora Marcelle Danelon
Na vida sempre encontramos pessoas especiais, pessoas que se
destacam e acabam se tornando grandes referenciais. Você foi quem me
apoiou em todos os momentos, me ensinando com toda sua excelência,
delicadeza e destreza.
Você é um exemplo de vida, de pessoa e acima de tudo de
profissional. Uma profissional completa, apaixonada pelo que faz, sempre
mostrando o lado bom das coisas. Nesse período aprendi muito com você,
não só Odontologia, metodologias, mas aprendi com a sua história, com os
seus conselhos, os quais foram fundamentais para mim nesse grande ciclo.
Eu desejo a você toda a felicidade do mundo, que Deus possa te
iluminar, e te guiar. E que todos os seus sonhos e objetivos sejam
realizados! Conte sempre comigo!
Obrigado por sempre acreditar em mim...
Meu eterno carinho e gratidão a você.
Aos meus amigos e companheiros da Pós-graduação,
Obrigado a vocês que de alguma forma contribuíram comigo nesse
período. Vou sentir muita saudade de todos! O meu desejo é que todos
alcancem seus objetivos.
José Guilherme Neves
Aos queridos alunos e amigos Francyenne, Gabriel, Sara e
Ronaldo.
Vocês me ensinaram muito nesse período. Me ensinaram sobre
determinação, capacidade e esforço. Ter alunos como vocês, com o
potencial de vocês, é algo desejado por qualquer pós-graduando e
professor.
Obrigado por todas as vezes que vocês me fizeram rir, por todos os
momentos em congressos e jornadas.
Vocês são muito especiais! Podem sempre contar comigo!
Ao aluno de Iniciação Científica Leonardo Raniel
Figueiredo,
Pela grande ajuda na realização da fase laboratorial deste trabalho, e
pela grande amizade!
Desejo a você todo o sucesso do mundo, que Deus realize todos os
seus sonhos e projetos. Eu só tenho a te agradecer por toda a ajuda, por
todos os dias que positivamente você chegava no laboratório pronto a
ajudar.
José Guilherme Neves
Aos docentes da Disciplina de Odontopediatria da
Faculdade de Odontologia de Araçatuba, UNESP,
Prof. Titular Célio Percinoto, Prof. Adj. Robson Frederico Cunha, Prof.
Ass. Dr. Juliano Pelim Pessan, Profa. Dra. Rosângela dos Santos Nery, Profa.
Adj. Sandra Maria Herondina Ávila de Aguiar, Profa. Ass. Dra. Cristiane
Duque, pela agradável convivência e conhecimentos transmitidos.
A Faculdade de Odontologia de Araçatuba,
Na pessoa dos professores: Prof. Titular Wilson Roberto Poi,
digníssimo Diretor e Prof. Titular João Eduardo Gomes Filho, digníssimo
Vice-Diretor.
Aos
funcionários
do
Departamento
de
Odontopediatria,
Obrigado a vocês que sempre nos receberam com tanto carinho,
cuidado e atenção. Obrigado por se preocuparem até com um simples café,
que nos faria despertar quando estávamos cansados.
Que Deus os abençoe sempre!
Ao Curso de Pós-Graduação em Ciência Odontológica
da Faculdade de Odontologia de Araçatuba-UNESP,
José Guilherme Neves
À Valéria, Cristiane e Lilian da Seção de Pós-Graduação
da Faculdade de Odontologia de Araçatuba-UNESP,
Pelo profissionalismo e atenção sempre carinhosa.
Ao Frigorífico FRIBOI
,
Pela permissão da coleta dos dentes bovinos.
A Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior
(CAPES) e a Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP)
–
Processo 2015/20829-5,
Pela concessão de recursos a mim e ao aluno de iniciação Científica
Leonardo Raniel Figueiredo.
José Guilherme Neves
José Guilherme Neves
“Não tenho palavras pra agradecer Tua
bondade
Dia após dia me cercas com fidelidade
nunca me deixes esquecer
Que tudo o que tenho
Tudo o que sou
O que vier a ser
Vem de Ti Senhor’’
José Guilherme Neves
José Guilherme Neves NEVES, J. G. Energia livre de superfície do esmalte dentário tratado com
Hexametafosfato de sódio, Cálcio e Fosfato. 2016 64f. Dissertação (Mestrado
em Ciência Odontológica, área de concentração Biomateriais) - Faculdade de
Odontologia de Araçatuba,Universidade Estadual Paulista, Araçatuba 2016.
Objetivo: Esse estudou avaliou a capacidade de adsorção do hexametafosfato de
sódio (HMP) em diferentes concentrações no esmalte dentário. Desenho: Blocos
de esmalte bovino (4 mm x 4 mm, n=144, 12/grupo) foram selecionados e
divididos em 12 grupos: 0%; 0,25%; 0,5%; e 1% HMP, e essas concentrações de
HMP seguida da aplicação em solução contendo Ca ou Ca-PO4 Os tratamentos
foram realizados por 1 min (1 mL/bloco), e a seguir expostos ao ar para que
houvesse a formação de película. A energia livre de superfície (mN/m) foi
calculada pela medida dos ângulos de contato de três líquidos sondas: água
deionizada, diiodometano e etileno glicol; determinando os componentes polar e
apolar da superfície do esmalte. Analisaram-se as concentrações de cálcio (Ca),
fosfato (PO4) e HMP nas soluções, antes e após os tratamentos. Os dados
apresentaram distribuição normal (Kolmogorov-Smirnov) e homogênea (Cochran)
e a seguir foram submetidos a ANOVA seguido pelo teste Student-Newman
Keuls (p<0,05). Resultado: Quanto maior a % de HMP nas soluções maior a
adsorção de HMP e a eletronegatividade na superfície do esmalte (p<0,05). A
adsorção de Ca foi maior com o aumento da % de HMP na solução (p<0,05)
reduzindo a eletronegatividade na superfície do esmalte. Maior adsorção de Ca e
PO4 ocorreu com 0,5% HMP e 1% HMP após o tratamento com solução Ca-PO4,
deixando a superfície menos eletronegativa quando comparado aos demais
José Guilherme Neves de esmalte mais eletronegativa favorecendo uma maior adsorção dos íons Ca e
PO4.
José Guilherme Neves
José Guilherme Neves NEVES, J.G. Surface free energy of enamel treated with sodium
hexametaphosphate, calcium and phosphate. 2016 64f. Dissertação
(Mestrado em Ciência Odontológica, área de concentração Biomateriais) -
Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista,
Araçatuba 2016.
Objective: This study evaluated the sodium hexametaphosphate (HMP) capacity
adsorption at different concentrations in the dental enamel. Design: Bovine
enamel blocks (4 mm x 4 mm, n = 144, 12 group) were selected and divided into
12 groups: 0%; 0.25%; 0.5%; and 1% HMP, and these HMP concentrations
followed by the application solution containing Ca or Ca-PO4. The treatments
were performed for 1 min (1 ml /block), and then exposed to air so that there was
the formation of a pelicle. The surface free energy (mN/m) was calculated by
measuring the contact angles of liquids three probes: deionized water,
diiodomethane and ethylene glycol; determining the polar and nonpolar
components of the enamel surface. It was analyzed the calcium (Ca), phosphate
(PO4) and HMP in the solutions, before and after treatment. The data were
normally distributed (Kolmogorov-Smirnov) and homogeneous (Cochran) and
then were subjected to ANOVA followed by Student-Newman Keuls test (p
<0.05). Results: The greater HMP % in solutions, the greater adsorption of HMP
and electronegative surface enamel (p<0.05). The Ca adsorption was higher with
HMP % increasin in the solution (p<0.05) reducing enamel surface
electronegativity. Increased adsorption of Ca and PO4 occurred in 0.5% and 1%
HMP and HMP after treatment with Ca-PO4 solution, leaving the less
electronegative surface when compared to the other treatments (P<0.05).
José Guilherme Neves electronegative surface enamel (p<0.05). The Ca adsorption was higher with
increasing HMP % in the solution (p<0.05) in reducing electronegativity enamel
surface. Increased adsorption of Ca and PO4 occurred in 0.5% and 1% HMP
HMP after treatment with Ca-PO4 solution, leaving the less electronegative
surface when compared to the other treatments (P <0.05). Conclusion: The HMP
promotes more electronegative in enamel surface, achieving greater adsorption of
Ca and PO4 ions.
José Guilherme Neves
José Guilherme Neves
LISTA DE FIGURAS
Figure 1. (A) Surface free-energies and their components (ϒSLW: Lifshitz-van der
Waals surface tension component; ϒSAB: Lewis acid-base interaction) with different enamel-surface treatments. (B) Influence of the treatments on the component polar of surface free energy on enamel surface: Lewis-acid (ϒS+) and Lewis-base (ϒS). Values denote mean and standard deviation (n = 12). Distinct letters show significant differences among mean considering % HMP and treatment or not with Ca and Ca and PO4 solution.
Figure 2. Mean (SD) of HMP adsorbed (A) on enamel surface after treatment
José Guilherme Neves
José Guilherme Neves
Table 1: Surface free energy and component values in test liquids
Table 2: Means (SD) of the contact angles ( °) of probing liquids and surface
José Guilherme Neves
José Guilherme Neves
LISTA DE ABREVIATURAS
ANOVA Análise de variância Ca2+ íon cálcio
CaCl2 Cloreto de Cálcio
CaF+ íon fluoreto de cálcio
HMP Hexametafosfato de sódio
HCl Ácido Clorídrico
H2O Água
LWAB Equação Ácido-base Lifhitz van der-Walls
mm Milímetro
mmol Milimol
ml Mililitro
min Minuto
mN/m MiliNilton por metro
NaOH Hidróxido de sódio
n Numero da amostra
sec Segundos
µL Microlitros
ϒ+ Componente receptor
ϒ- Componente doador
ϒLW Componente van der Walls
ϒAB
Componente Ácido-Base de Lewis
ϒL
José Guilherme Neves
José Guilherme Neves
SUMÁRIO
ABSTRACT 35
INTRODUCTION 36
MATERIALS AND METHODS 38
RESULTS 41
DISCUSION 42 ACKNOWLEDGMENTS 46 REFERENCES 46
ANEXOS 58
José Guilherme Neves
34
Surface free energy of enamel treated with sodium hexametaphosphate, calcium and phosphate
J.G. Nevesa, M. Danelona, L.R. Figueiredoa, J.P. Pessana, A.C.B. Delbema
aAraçatuba Dental School, Univ. Estadual Paulista (UNESP)
Department of Pediatric Dentistry and Public Health
Rua José Bonifácio 1193 Araçatuba, SP - Cep 16015-050 – Brazil
Short title: Surface free energy of enamel treated with sodium hexametaphosphate
Corresponding author:
Alberto Carlos Botazzo Delbem São Paulo State University – UNESP
Department of Pediatric Dentistry and Public Health Rua José Bonifácio 1193
16015-050 Araçatuba – SP - Brazil Tel. +55 18 3636 3235
Fax +55 18 3636 3332
Email: adelbem@foa.unesp.br
*De acordo com as instruções aos autores do periódico Caries Research.
(Anexo A)
35
ABSTRACT
Objective: This study evaluated the sodium hexametaphosphate (HMP) capacity
adsorption at different concentrations in the dental enamel. Design: Bovine
enamel blocks (4 mm x 4 mm, n = 144, 12/group) were selected and divided into
12 groups: 0%; 0.25%; 0.5%; and 1% HMP, and these HMP concentrations
followed by the application solution containing Ca or Ca-PO4. The treatments
were performed for 1 min (1 ml/block), and then exposed to air so that there was
the formation of a pelicle. The surface free energy (mN/m) was calculated by
measuring the contact angles of liquids three probes: deionized water,
diiodomethane and ethylene glycol; determining the polar and nonpolar
components of the enamel surface. It was analyzed the calcium (Ca), phosphate
(PO4) and HMP in the solutions, before and after treatment. The data were
normally distributed (Kolmogorov-Smirnov) and homogeneous (Cochran) and
then were subjected to ANOVA followed by Student-Newman Keuls test (p
<0.05). Results: The greater HMP % in solutions, the greater adsorption of HMP
and electronegative surface enamel (p<0.05). The Ca adsorption was higher with
HMP % increasin in the solution (p<0.05) reducing enamel surface
electronegativity. Increased adsorption of Ca and PO4 occurred in 0.5% and 1%
HMP and HMP after treatment with Ca-PO4 solution, leaving the less
electronegative surface when compared to the other treatments (P<0.05) Results:
The higher HMP% solutions was greater HMP adsorption and electronegative
surface enamel (p<0.05). The Ca adsorption was higher with increasing HMP %
in the solution (p<0.05) in reducing electronegativity enamel surface. Increased
adsorption of Ca and PO4 occurred in 0.5% and 1% HMP HMP after treatment
36
José Guilherme Neves the other treatments (P<0.05). Conclusion: The HMP promotes more
electronegative in enamel surface, achieving greater adsorption of Ca and PO4
ions.
Keywords: Phosphates, Dental enamel, Energy.
INTRODUCTION
Cyclophosphates have been added to fluoride products as toothpastes [da
Camara et al., 2014; da Camara et al., 2015; de Castro et al., 2015; Danelon et
al., 2015], gels [Danelon et al., 2013; Danelon et al., 2014; Pancote et al., 2014;
Conceição et al., 2015], varnishes [Moretto et al., 2013; Manarelli et al., 2013;
Manarelli et al., 2014] and mounthrinses [Manarelli et al., 2011; Favretto et al.,
2013] with propose to increase the products capacity to reduce teeth
demineralization and enhance their remineralization. As dental caries is the most
common chronic oral disease in worldwide [Clarkson, 2010], increase the ability
of fluoride products in reduce the mineral loss can improve the effect against
dental caries. Sodium hexametaphosphate (HMP) is one of cyclic inorganic
phosphate [Kulaev et al., 2005] that reduces enamel desmineralization [da
Camara et al., 2014; da Camara et al., 2015; Conceição et al., 2015; da Camara
et al., 2016] due to its ability to adsorption on enamel surface and increase
permselectivity, facilitating the diffusion of cations into the enamel [McGaughey
and Stowell, 1977; van Dijk et al., 1980]. The assumption would be that the HMP
would retain ionic species like CaF+ and Ca2+, leading to a reticular formation of
37
José Guilherme Neves re-mineralization processes [da Camara et al., 2014; da Camara et al., 2015;
Conceição et al., 2015]. However, this hypothesis needs to be proven as HMP is
a negatively charged cyclic phosphate [Choi et al., 1993] as well as the enamel
[McGaughey and Stowell, 1977; van Dijk et al., 1980]. Thus, the study of the
interactions and chemical bonds among HMP, enamel surface and rich medium in
calcium phosphate can help us to understand and to prove the above hypothesis.
The surface reactivity is evaluated through the surface free energy (SFE)
[O`Brien, 1997] measuring the contact angle formed by different liquids on a solid
surface [Zisman, 1964]. Among the different methods to achieving these
parameters [Zisman, 1964; Owens, 1969; Neuman, 1974; Baier, 1992], the
approach described by Van Oss, Fowkes and Good and Chaudhury provides the
most complete SFE description. It allows calculating the dispersion values,
acid-base Lewis parts, polar force and hydrogen bond force, determining the Lifshitz–
van der Waals acid–base equation (LWAB) [Fowkes, 1987; van Oss, 1987; van
Oss, 1988; Good, 1992; Chaudury, 1996]. The dispersion force discusses
interactions values of nonpolar molecules, the polarity force shows the
interactions of metal molecules and the hydrogen bond strength reports the
interaction of water and hydroxyl groups [Comyn, 1992; Ueta, 2016]. This method
has the same foundation of hydrophobicity angle measurement, but
hydrophobicity is measured only by angles made with water, different from this
proposal where is used at least three liquids with different physico-chemical
properties [Blunden, 1994; Carlen, 2001; Sipahi, 2001].
Therefore, the aim of this study was to analyze the HMP adsorption and
38
José Guilherme Neves if the HMP treatment would interfere in the adsorption of calcium phosphate on
enamel. The null hypothesis was that the HMP is not adsorbed and do not alters
the SFE of the enamel and the adsorption of calcium phosphate.
MATERIAL AND METHODS
Experimental design
Enamel blocks (4 x 4 mm, n= 144) of bovine incisors were stored in
formaldehyde solution 2% for 30 days. The enamel surfaces of blocks were
sequentially polished, and as inclusion criteria the blocks should be flat, with no
risk, cracks or hypoplasia. They were divided in twelve groups (n = 12/group) and
immersed in the solutions content: free of HMP (0%), 0.25% HMP, 0.5% HMP
and 1% HMP with or without posterior treatment with calcium (1.25 mmol/L Ca) or
calcium and phosphate solutions (1.25 mmol/L Ca and 3.5 mmol/L P). Surface
free energy analysis was performed trough contact angles, and of calcium and
phosphorus concentrations determined before and after treatments.
Formulation of the solutions and treatment
Solutions were prepared using microparticulate HMP (Aldrich Chemistry,
UK), in concentrations of 0%, 0.25%, 0.5% and 1%. Solutions containing calcium
(1.25 mmol/L CaCl2·2H2O) and calcium and phosphate (1.25 mmol/L CaCl2·2H2O
and 3.5 mmol/L NaH2PO4·2H2O; Sigma, USA) also was prepared [Fejerskov,
2003]. Blocks (n = 12/group) were immersed in individual flasks containing 1 mL
39
José Guilherme Neves Brazil) over a period of 2 min. After this treatment, the blocks were washed with
deionized water for 30 seconds and dried. Twelve blocks of each group were
stored to posterior analysis, another 12 were immersed in an individual flask
containing 1 mL of a calcium solution and the last 12 blocks immersed in a
calcium/phosphate solution, under constant agitation for 1 min.
Calcium and phosphorus analysis
The Ca analysis was performed by a spectrophotometric (Microplate
Spectrophotometer EON, Biotek, USA), with a wavelength of 650 nm by adopting
a colorimetric method Arsenazo III [Vogel et al., 1983]. Aliquots of 5 µL were
taken from the samples and 50 µL of deionized water and arsenazo were added.
For calibration, standards containing 40 to 200 µg Ca/mL were used. The
phosphorus was colorimetric measured by the molybdate method through an
aliquot of 20 µL from the samples and in sequence, added to a mixture of 50 µL
of molybdate and 20 µL reactive reducer, as described by Fiske and Subbarow
(1925). This analysis was performed before and after the treatments and the
adsorption to enamel calculated from the initial concentrations of these
compounds in the solutions and the concentration after treatment (, µg). The
phosphorus of the HMP solutions was dosed after acid hydrolysis. Aliquots of
0.2 mL of the HMP solutions were added of 0.2 mL of hydrochloric acid (HCl) 1.0
40
José Guilherme Neves
SFE Measurements
The physico-chemical properties of the enamel surface were characterized
by contact angle measurements, using the sessile drop method to determine the
surface free energy. Measurements were performed by an automatic goniometer
(DSA 100S, Krüss, Hamburg, Germany) using three liquid contained within of
probes: diiodomethane, water and ethylene glycol (Table 1). The treated blocks
were exposed for 45 min in the air in order to stabilize the formed pellicle [van der
Mei et al., 2002], after this 0.5 µL of each liquid has been dispensed on the blocks
surface, and the contact angles were measured using images captured by a CCD
camera. For each specimen 5 measurements were performed at 20° C [Harnett
et al., 2007; van der Mei et al., 2002]. Different parameters, such as acid (+,
donor component), base (-, receptor component) and Lifshiz van der Waals (LW,
nonpolar component) of surface free energy (mN/m) were calculated according to
the model of van Oss, Chaudhery and Good for the determination of the
interaction free energy substrates [Van Oss et al., 1990; Della Volpe, 1997].
Statistical Analysis
The data presented a normal (Kolmogorov-Smirnov) and homogeneous
(Cochran) distribution and were submitted to analysis of variance (two-way)
followed by Student-Newman-Keuls test for multiple comparisons. Analyses were
performed using the Sigma Plot statistical software (version 12.0) with a
significance level set at 5 %. The values of contact angle, SFE parameters, HMP,
41
José Guilherme Neves HMP and post treatment with calcium and calcium/phosphate solutions as the
variation factor.
RESULTS
Treatments with HMP led to lower values of contact angles for three
probes liquids (p<0.001) compared to no-HMP (Table 2), which means that HMP
effectively affects the surface composition. Surface free energy (S) was only
higher with 0.25 % HMP (p=0.002). The values of apolar component (SLW) from
enamel surface were higher after treatment with HMP (p=0.001). The values of
polar component (SAB = Lewis acid-base) became more negative with the
increases of %HMP (p<0.017) (Figure 1). Among the parameters from SAB, S+ =
eletron-acceptor (Lewis acid) and S- = eletron-donor (Lewis base), the S- values
got greater with the increases of %HMP (p<0.001). The same was observed with
the adsorption of HMP on enamel (p<0.001) (Figure 2). There was a correlation
between values of HMP adsorption and SAB (Pearson’s r = 0.600) and S
-(Pearson’s r = -0.736).
The enamel surface without pretreatment with HMP but treated with
calcium solution presented higher values of contact angles for probes liquids
(p<0.005) as well as a reduction of S values (p=0.013) when compared to
enamel without any treatment (Table 2). Furthermore, there was not alteration in
the SAB, S+ and S- values (p>0.119) and slightly reduction of SLW values
(p=0.002). The treatment with calcium on enamel surface pretreated with HMP
42
José Guilherme Neves calcium treatment. The S values were higher for enamels treated with 0.25% and
0.5% of HMP (p<0.002). Calcium treatment led to less negatives SAB values
according to HMP content and a reduction of 57% for S- values for all % of HMP
(p<0.001). The values of SLW post treatment with calcium were lower in relation to
its correspondent % of HMP without calcium treatment (p<0.013).
The treatment with Ca-PO4 solution on enamel surface pretreated with
HMP showed higher contact angles for probes liquids compared to enamel
treated solely with HMP. The S values were higher for enamel treated with 0.5%
and 1% of HMP (p<0.002). Larger adsorption of HMP on enamel increased
absorption of calcium and phosphate after treatment with Ca-PO4 solution. There
was dose-response relationship between adsorption of HMP and Ca+ (Pearson’s
r = 0.601) and PO4 (Pearson’s r = 0.622) adsorption on enamel. As consequence,
the SAB values became positive with the increases of calcium (Pearson’s r =
-0.637) and phosphate (Pearson’s r = -0.621) adsorption on enamel. Also, S
-values decreased (p<0.002) showing correlation with calcium (Pearson’s r =
0.759) and phosphate (Pearson’s r = 0.712) adsorption on enamel.
DISCUSSION
This study evaluated the SFE alteration on enamel after treatment with
HMP and the calcium and phosphate interference. The results showed that the
adsorption of HMP on enamel enhanced the adsorption of calcium and
phosphate. Thus, the null hypothesis was rejected. A dose-response relationship
43
José Guilherme Neves adsorption, polar component (SAB = Lewis acid-base) and S (Lewis base
eletron-donor).
Considering only the contact angle with water (W), it is possible to affirm
that the treatment with HMP increases the hydrophilic character of enamel
surface, which makes it more liable to wetting, given that the contact angles were
lower than 65° [Vogle, 1998]. The surface free energy (S) and its Lifshiz van der
Waals (LW) and Lewis acid-base (AB) components also indicate that enamel
surface treated with HMP are more susceptible to bacterial adhesion [Rüttermann
et al., 2011; Rüttermann et al., 2014; Tiznado-Orozco et al., 2015].
The enamel prior to HMP treatment showed hydrophobic character and
lesser probability to bacterial adhesion inasmuch as S < 30 mN/m [Knorr et al.,
2005; Rüttermann et al., 2011] and S+ close to zero, as well as S is less than
28.5 mN/m [van Oss, 1993; van Oss, 1995]. Nevertheless, attractive interaction to
biological species, compounds and cells was present but weaker than in enamel
treated with HMP because SLW was higher than 22 mN/m [van Oss, 1993; van
Oss, 1995]. In this case, S is lower because of lower SAB values indicating a
surface less susceptible to cell adhesion [Knorr et al., 2005; Lee et al., 2009]. The
adsorption of HMP on enamel, on the other hand, occur leaving the surface
negatively charged. Despite S values higher than 28.3 mN/m that characterizes
a hydrophilic surface [van Oss, 1993; van Oss, 1995], the S remained the same
because a negative SAB even with higher and positive SLW values due the HMP
44
José Guilherme Neves cell adhesion, data from W, SLW and SAB (S+; S) effectively indicate a surface
that favors the adherence of biofilms [Olsson et al., 1992; Quirynen et al., 1989].
As the surface enamel became negatively charged due HMP the
adsorption of HMP, the adsorption of calcium on enamel is enhanced in the same
ratio as the S parameter increases. Negatively surfaces can be partly or wholly
neutralized by plurivalent cations leading to a hydrophobic surface [van Oss,
1995]. Apolar and polar components from the surface free energy were lower and
close to enamel prior to HMP, which confirms previous studies about the
reduction of enamel demineralization due the adsorption of HMP and its bond to
calcium on enamel surface [da Camara et al., 2014; da Camara et al., 2015;
Conceição et al., 2015]. Enamel without treatment adsorbed around 200% less
calcium that surfaces treated with HMP. It indicates no binding to phosphate ions
on the hydroxyapatite surface [Harding et al., 2005; Vandiver et al., 2005] since
the values of polar component (SAB) and its parameters (S+; S) did not change.
To calcium be adsorbed without changing the surface charge probably there was
a displacement of two surface H+ ions by a Ca2+ ion [Harding et al., 2005].
Nevertheless, the surface became more hydrophobic because of higher W and
lower S (as consequence of SLW) when compared to enamel prior to HMP or
calcium. Thus, the formation of HMP layer covering the enamel gives more
phosphates sites increasing the retention of calcium. As consequence, it occurs a
supersaturation these ions close to surface with possibility of higher adsorption of
phosphates ions.
The treatment of the enamel surface prior to HMP with calcium phosphate
45
José Guilherme Neves even reducing S and SLW values. As the calcium phosphate solution has the
possibility to form ionic species with different charges and activities, enamel
reactivity depends of the electrical charge created on the enamel surface.
Utilizing the PHREEQC Interactive speciation software (version 2.18.3.5570) to
calculate these species, it was possible to arrange in a descending order of
reactivity, from H2PO4 > Ca2 > HPO42 > CaH2PO4 at 37°C, pH 5.92, and
density of 1.0 g/cm3. The adsorption of Ca2 and HPO42 seems to explain the
increase of the negative charges (S = 40.3 mN/m) on enamel surface prior to
HMP faced with lower adsorption values. As the enamel surface present negative
charges after the treatment with HMP, the reactivity with Ca2 is greater leading to
more adsorption of H2PO4 and CaH2PO4, resulting in higher SAB values and
reduction in the S parameter. These differences might explain the higher ability
to reduce the demineralization or enhance the remineralization of enamel adding
HMP oral health products [da Camara et al., 2014; da Camara et al., 2015;
Conceição et al., 2015]. Although it was possible to determine the influence of
HMP in the adsorption of important ions in the process of des-remineralization of
enamel, the role played by fluorite in a such system still undetermined.
It was concluded that the adsorption of HMP on enamel increased the
adsorption of calcium and phosphate. The polar component (SAB) of the surface
free energy and its S parameter from the enamel surface were higher with the
46
José Guilherme Neves
ACKNOWLEDGMENTS
This study was supported by CAPES (Brazilian Coordination ofTraining of Higher Education Graduate) and FAPESP (The State of São Paulo Research Foundation, 2015/20829-5) for the concession of a scholarship to the four author.
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Table legend
Table 1. Surface free energy and component values in test liquids
Table 2. Means (SD) of the contact angles ( °) of probing liquids and surface
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Figure legends
Figure 1. (A) Surface free-energies and their components (SLW: Lifshitz-van der Waals surface tension component; SAB: Lewis acid-base interaction) with different enamel-surface treatments. (B) Influence of the treatments on the component polar of surface free energy on enamel surface: Lewis-acid (S+) and Lewis-base (S). Values denote mean and standard deviation (n = 12). Distinct letters show significant differences among mean considering % HMP and treatment or not with Ca and Ca and PO4 solution (Student-Newman-Keuls, p <0.05).
Figure 2. Mean (SD) of HMP adsorbed (A) on enamel surface after treatment
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Table 1. Surface free energy and component values in test liquids
Liquid CAS. Manufacturer L LLW LAB L+ L-
Deionized water 72.8 21.8 0.00 25.50 25.50
Diiodomethane 75116 Sigma-Aldrich 50.80 50.42 0.38 0.00 0.00
Ethileneglycol 107211 Sigma-Aldrich 48.0 29.0 18.9 3.0 30.10
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José Guilherme Neves
Table 2. Means (SD) of the contact angles ( °) of probing liquids and surface
free energy (S) after treatment of enamel surface with HMP and after treatment with Ca or Ca and PO4 solution on enamel surface pretreated with HMP solutions (n = 12)
Treatments Water (°) Diiodomethane (°) Ethylene glycol(°) (mN/m) S
no-Ca and PO4
0%HMP 67.7(4.1) a 52.4(5.5) a 55.4(4.2) a* 28.8(4.6) a
0.25%HM
P 48.0
b
(4.4) 39.0
b
(5.1) 37.5
b
(5.7) 33.6
b* (3.2)
0.5%HMP 43.1(3.8) c 48.0(6.4) c* 40.8(4.3) b 27.5(3.9) a
1%HMP 40.3(8.6) c 43.6(5.4) d 40.8(5.1) b 25.9(5.2) a*
Ca
0%HMP 73.6(6.3) a 57.6(2.6) a* 62.1(3.7) a 25.3(2.3) a*
0.25%HM
P 68.5
a*
(6.0) 55.8
a*
(3.9) 53.4
b*
(2.9) 31.5
b*
(3.4)
0.5%HMP 63.7(5.6) b 50.7(3.4) b* 49.7(4.3) c 31.7(3.4) b*
1%HMP 69.4(6.2) a* 54.2(3.5) a* 58.5(3.5) d 26.5(2.9) a*
Ca-PO4
0%HMP 58.4(3.1) a 58.3(2.8) a* 54.7(3.2) a* 24.0(4.2) a*
0.25%HM
P 64.8
b*
(2.2) 55.0
a,b*
(2.3) 54.4
a*
(2.5) 27.3
b (3.4)
0.5%HMP 68.8(3.1) b,c 56.7(3.1) a,b 53.8(3.3) a 31.0(3.0) c*
1%HMP 70.4(3.7) c* 53.4(2.4) b* 52.2(3.1) a 33.2(2.3) c
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José Guilherme Neves
56
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Figure 2. Mean (SD) of HMP adsorbed (A) on enamel surface after treatment
57
José Guilherme Neves
58
ANEXO A
INSTRUÇÕES AOS AUTORES
Guidelines for Authors
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José Guilherme Neves
ANEXO B
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José Guilherme Neves
ANEXO C
TRATAMENTO DOS BLOCOS DE ESMALTE COM
SOLUÇÕES EXPERIMENTAIS
30
1 min Mesa Agitadora
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José Guilherme Neves
ANEXO D
ANÁLISE DA ENERGIA LIVRE DE
SUPERFICIE DO ESMALTE DENTÁRIO
Goniômetro Automático DAS110S, Kruse,
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José Guilherme Neves
ANEXO E
DOSAGEM DE CÁLCIO NAS SOLUÇÕES
EXPERIMENTAIS
5 µL da amostra, 50 µL de Água deionizada,
50 µL de Arsenazo III
Espectrofotômetro de microplaca EON, Biotek,
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José Guilherme Neves
ANEXO F
DOSAGEM DE HMP NAS SOLUÇÕES
EXPERIMENTAIS
200 µL da solução HMP +
200 µL de HCl 1 M
Banho Térmico TE- 054 MAG, Tecnal
20 µL da amostra +
50 µL de molibidato
+
20 µL de reativo redutor
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José Guilherme Neves
ANEXO G
DOSAGEM DE FÓSFORO NAS SOLUÇÕES
20 µL da amostra +
50 µL de molibidato
+
20 µL de reativo redutor
Espectrofotômetro de microplaca EON,
